Affinipure goat anti mouse igg h l

IPMA was used to assess the virus titration as previously described^{48 (link)}. Briefly, cells were fixed with 100 μl of 4% cold formalin for 15 min at room temperature (RT). The fixed cells were washed with 100 μl phosphate-buffered saline (PBS) once after that and twice with 100 μl of 0.5% PBS Tween-20 (PBST) and then blocked with 100 μL of 1% bovine serum albumin (BSA) in 0.5% PBST for 30 min at RT. Then, the cells were washed. Seventy microliters of anti-PRRSV NC protein monoclonal antibody (Median Diagnostics, Gangwondo, Korea) diluted at a ratio of 1:400 was used to stain cells at RT for 60 min. The cells were washed and incubated again with 50 μl of peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson Immuno Research, Pennsylvania, USA) at a dilution of 1:600 for 60 min at RT, washed and counterstained with 1,5-diaminopentane (DAP) substrate for 5 min, washed with distilled water and examined under a microscope. Virus titer was determined using the Reed–Muench method^{46 (link)}, also expressed as TCID₅₀, described as diluting a virus required to infect 50% of a given cell culture^{49 (link)}.

Leukocytes (1 × 10⁶ cell/mL) from kidney, liver, spleen, and PBL from three olive flounders (62.3 ± 9.6 g at 16 °C) were prepared by centrifugation at 500 × g for 3 min according to Section 2.6. For flow cytometry analysis, leukocytes were incubated with respective CD marker mAbs- 4B2 (anti-CD3), 10F8-3 (anti-CD4-1) and 1D3 (anti-CD4-2) for 1 h at RT (room temperature, 25 °C), respectively [26 (link),27 (link),28 (link)]. This was followed by fluorescein isothiocyanate (FITC)-conjugated AffiniPure goat antimouse IgG (H + L; Jackson ImmunoResearch, Pennsylvania, USA) staining with a 1:200 dilution for 30 min at 37 °C. Negative controls were treated with FITC conjugate only. Leukocytes were washed twice with 1× PBS between each step, re-suspended in 1× PBS, and analyzed using a FACS Calibur™ (BD biosciences, San Jose, USA) flow cytometer, measuring at least 10,000 events per sample.

Whole-cell lysates were prepared in lysis buffer (10 mM Tris-HCl, pH 8, 140 mM NaCl, 5 mM EDTA, 0.025% NaN₃, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM PMSF, 5 μg ml^–1 leupeptin, 2 μg ml^–1 aprotinin, 50 mM NaF, and 1 mM Na₂VO₃), after which the lysates (40–50 μg) were subjected to SDS-PAGE. Then proteins were transferred to PVDF membranes (Millipore) and incubated with the primary antibody, followed by incubation with horseradish peroxidase-conjugated immunoglobulin G and detection by enhanced chemiluminescence (GE). The primary antibodies for western blotting were anti-p53 antibody (DO-1) (Santa Cruz Biotechnology, sc-126), anti-p53 antibody (Leica, NCL-L-p53-CM5p), and anti-PAN-actin antibody (Cell Signaling Technology, 4968S). All primary antibodies were used at a dilution of 1:1000. The secondary antibody for anti-p53 antibody (sc-126) was peroxidase-conjugated AffiniPure goat anti-mouse IgG(H+L) (Jackson Immunoresearch, 115-035-003), while peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (Jackson Immunoresearch, 111-035-003) was used for anti-p53 antibody (NCL-L-p53-CM5p) and anti-PAN-actin antibody (4968S).

Glycopeptides were diluted in 0.05 M NaHCO₃, pH 9.6, coated at a concentration of 1 μg/mL in a volume of 100 μL/well to 96-well MaxiSorp plates (Nunc, Roskilde, Denmark) and incubated at 4 °C overnight. Plates were washed three times with PBS containing 0.05% (v/v) Tween (PBST) using a 405TS Miroplate Washer (BioTek, Winooski, VT) and then blocked with a volume of 200 μL/well of 3% (w/v) BSA/PBS at room temperature for two hours. Plates were washed three times with PBST and then incubated with primary antibodies or isotype control antibodies diluted in 1% (w/v) BSA/PBS to a concentration of 0.25 μg/mL (Ma695 0.5 μg/mL) and using a volume of 50 μL/well at room temperature for two hours. Plates were washed three times with PBST and then incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted 1:10000 in 1% (w/v) BSA/PBS using a volume of 100 μL/well at room temperature for one hour. After plates were washed six times with PBST, Super AquaBlue ELISA Substrate (Thermo Fisher Scientific, Waltham, MA) was added using a volume of 100 μL/well and incubated at room temperature for 35 minutes to visualise antibody binding. Absorbance was measured at a wavelength of 405/492 nm by a Multiscan FC spectrophotometer (Thermo Fisher Scientific). Values were generated in duplicates and repeated at least two times.